The present invention relates to a method for thorough mixing of liquids in microcavities and a device for carrying out said method.
Microcavities, for example in an arrangement of microtitre plates, are employed in pharmaceutical research and diagnostics as reaction vessels. On the basis of the standard format of microtitre plates highly automated processing sequences are possible in modern laboratories. For example, pipetting robots, units for optical reading of biological assays and also the corresponding transport systems are thus matched to the standard format. Such standard microtitre plates exist currently with 96, 384 or 1536 cavities. Typical volumes per cavity are in the range of 300 μl for 96 titre plates, approximately 75 μl for 384 microtitre plates and approximately 12 μl for 1536 titre plates. Microtitre plates are generally made from plastic, for example polypropylene or polystyrol, and are frequently coated or biologically functionalised.
Miniaturising in the form of such microtitre plates or respectively microcavities is generally based on often expensive reagents and in the fact that sample material is frequently not available in the desired quantity, so that reactions at high sample concentration can be carried out only if the volumes are accordingly reduced.
So as to accelerate the reactions and also to ensure homogeneous reaction conditions, it is desirable to mix the reactants during the reaction. This is of significance in particular whenever a reaction partner (“sample”) is bound, that is, an inhomogeneous assay is present. Here, thorough mixing can prevent depletion of the sample on the bound probes. In the case of insufficiently thorough mixing frequently diffusion of the reactants quite generally is the time-determining step. This results in long reaction times and minimal sample throughput.
Microtitre plates or respectively in general microcavities are mixed thoroughly in known methods by means of so-called agitators. Such agitators comprise mechanically mobile components and are in part difficult to integrate into highly-automated lines. The thorough mixing is also highly inefficient in particular in small cavities, therefore for example 384 microtitre plates or 1536 microtitre plates. With such small microcavities small quantities of liquid are seemingly highly viscous and only laminar currents in small volumes are possible, that is, there is no turbulence which might cause effective thorough mixing. To achieve an adequate mixing effect, despite the viscosity becoming seemingly high in small quantities of liquid, a high output from the agitator is required.
WO 00/10011 thus describes a method, by means whereof a microcavity in the frequency range from 1 to 300 kHz is agitated. Outputs of 0.1 to 10 Watt are applied.
The literature describes other different methods for thoroughly mixing small quantities of liquid.
US 2002/0009015 A1 describes the use of cavitation for mixing, therefore nucleation, expansion and disintegration or collapse of a local vacuum space in the liquid or a bubble, therefore a local gas/steam space in the liquid, based on an acoustic pressure field. Mixing the liquid is achieved by the intrinsic dynamics of the local vacuum space or respectively the bubble, therefore its expansion and disintegration. To lower the acoustic output threshold for forming the local vacuum spaces or respectively bubbles, nucleation nuclei are needed. These nucleation nuclei heighten the danger of contamination. In addition to this, the development of local vacuum spaces or bubbles is often unwanted.
Other known method (for example “Microfluidic motion generation with acoustic waves”, X. Zhu et al. Sensors and Actuators, A. Physical, Vol. 66/1-3, page 355 to 360 (1998) or “Novel acoustic wave micromixer”, V.Vivek et al., IEEE International Microelectro mechanical systems conference 2002, pages 668 to 673, or U.S. Pat. No. 5,674,742) describe the use of membranous elements, which oscillate in so-called “flexural plate wave modes”. The motion-compromising medium is at the same time in direct contact with the liquid. The manufacturing of such thin membranes is highly complicated and the danger of contamination by contact of liquid with the motion-compromising medium is heightened.
U.S. Pat. No. 6,357,907 B1 describes the use of magnetic spheres, moving in an external, temporally or spatially variable magnetic field. To carry out the mixing procedure the spheres must be introduced to the liquid, an action often not desired on account of contamination problems.
U.S. Pat. No. 6,244,738 B1 describes a mixing procedure in a long-stretched-out closed channel. Two liquid currents flow past an ultrasound sender and are intermixed in the microchannel. To carry out the method a complicated structure with a microchannel system is needed and no separate individual volumes can be mixed.
U.S. Pat. No. 5,736,100 describes the use of a rotary table with small vessels, in which microcavities, for example Eppendorf caps, can be set. In these caps there is for example water, which is radiated from the outside with ultrasound. The described device therefore works as a conventional ultrasound bath. The water is set in oscillating motion and acts as a motion-compromising element directly on each cap, which is agitated in this way.
DE-A-101 17 772 describes the thorough mixing of liquids using surface sound waves, generated by means of interdigital transducers. The liquid is directly on the sound-compromising medium itself. At least in the case of multiple use of the devices there is the danger of contamination. Use with a microtitre plate is not possible in the arrangements described.